Study of Mutations in the DNA gyrase gyrA Gene of Escherichia coli

Quinolones are a large and widely consumed class of synthetic drugs. Expanded-spectrum quinolones, like ciprofloxacin are highly effective against Gram-negative bacteria, especially Escherichia coli. In E. coli the major target for quinolones is DNA gyrase. This enzyme is composed of two subunits, GyrA and GyrB encoding by gyrA and gyrB, respectively. Mutations in either of these genes cause quinolone resistance. Mutations in QRDR section of gyrA are more common in quinolone resistant clinical isolates. However, a mutation outside of this region was also reported. Thus, this study was aimed to provide more information on mutations sites in gyrA. For this purpose, spontaneous ciprofloxacin resistant mutants arisen in cultures of E. coli ATCC 25922 and MG1655 were isolated on LB agar containing ciprofloxacin. Next, the MICs of these clones were measured and the presence of mutation in gyrA was investigated. Results showed that the frequency of ciprofloxacin resistant mutants in cultures of E. coli strains was low. However, these mutants had different MICs, depending on the day of isolation. Most of ciprofloxacin-resistant mutants possess mutations in QRDR region and precisely at Ser-83. However, mutations outside of this region were also found at Tyr-50 and Ala-119. In conclusion, the presence of mutations at amino acids 50 and 119 suggests that in addition to QRDR section and Tyr-122, these sites are also essential for DNA gyrase activity.

Copyright ? 2010 by School of PharmacyShaheed Beheshti University of Medical Sciences and Health Services

Original Article

Study of Mutations in the DNA gyrase gyrA Gene of
Escherichia coli

Razieh Pourahmad Jaktaji* and Ehsan Mohiti

Department of Genetics, Faculty of Science,
University of Shahrekord, Shahrekord, Iran.

Abstract

Quinolones are a large and widely consumed class of synthetic drugs.
Expanded-spectrum quinolones, like ciprofloxacin are highly effective against
Gram-negative bacteria, especially Escherichia coli. In E. coli the major target
for quinolones is DNA gyrase. This enzyme is composed of two subunits, GyrA and
GyrB encoding by gyrA and gyrB, respectively. Mutations in either of these genes
cause quinolone resistance. Mutations in QRDR section of gyrA are more common in
quinolone resistant clinical isolates. However, a mutation outside of this
region was also reported. Thus, this study was aimed to provide more information
on mutations sites in gyrA. For this purpose, spontaneous ciprofloxacin
resistant mutants arisen in cultures of E. coli ATCC 25922 and MG1655 were
isolated on LB agar containing ciprofloxacin. Next, the MICs of these clones
were measured and the presence of mutation in gyrA was investigated. Results
showed that the frequency of ciprofloxacin resistant mutants in cultures of E.
coli strains was low. However, these mutants had different MICs, depending on
the day of isolation. Most of ciprofloxacin-resistant mutants possess mutations
in QRDR region and precisely at Ser-83. However, mutations outside of this
region were also found at Tyr-50 and Ala-119. In conclusion, the presence of
mutations at amino acids 50 and 119 suggests that in addition to QRDR section
and Tyr-122, these sites are also essential for DNA gyrase activity.

Quinolones are a large and widely consumed class of synthetic drugs (1).
First-generation (acidic) quinolones, including nalidixic acid and oxolinic
acid, have been only used for treatment of urinary tract infections (2).
However, modification of subsequent generations has increased their spectrum and
potency. One of these modifications has been the addition of a fluorine atom at
position C-6 of drug molecules, for instance in ciprofloxacin (CFX), which leads
to wide potent activity against different Gram-negative bacteria (1). Moreover,
due to the presence of a secondary amine in addition to carboxylic acid found in
all members of family, CFX is a amphoteric quinolone rather than acidic one
(Figure 1).

Fluoroquinolones, such as CFX have been used to treat a great variety of
infections, including urinary tract infections, blood stream infection, enteric
infections or respiratory tract infections (3). Unfortunately, frequent use and
sometimes misuse of CFX leads to the emergence of CFX-resistant bacteria,
especially in Gram-negative bacteria such as E. coli (4). In E. coli, the major
target for quinolones is DNA gyrase (2, 5, 6). DNA gyrase is a tetrameric enzyme
composed of two A subunits and two B subunits encoded by gyrA and gyrB,
respectively (7, 8). This enzyme belongs to type II topoisomerase family which
is able to supercoil and to uncoil DNA helix by cleaving both strands of helix,
passing another segment of the helix through resulting double strand break (DSB)
and resealing this DSB in the expense of ATP hydrolysis (9). These activities
are essential in DNA replication, transcription and recombination. On the other
hand, there are some minor targets for quinolones in Gram-negative bacteria,
including parC and parE encoding subunits of topoisomerase IV, another member of
type II topoisomerase (2). Quinolones bind to gyrase-DNA complex, called
cleavable complex due to the presence of DSB, and form gyrase-quinolone-DNA
ternary complex (5, 6). Ultimate denaturation or disruption of gyrase in ternary
complex results in the generation of DSB and thereby replication blockage and
cell death (10, 11).

Mutations in either gyrA or gyrB cause quinolone resistance. (12, 13). However,
mutations in the gyrA gene are more common in quinolone-resistant clinical
isolates of E. coli (14, 15). DNA sequence analysis has shown that most of the
mutations have been located in the first half of gyrA gene in the region called
quinolone resistance determining region (QRDR) (12). This region is in close
relation with the active site of GyrA (Tyr-122), which interacts with DNA and
quinolone (16). However, a mutation outside this region was also reported (17).
To gain more information on mutations sites in gyrA, spontaneous CFX-resistant
mutants of pathogenic E. coli ATCC 25922 and non pathogenic one, MG1655 were
isolated on LB agar containing CFX.

E. coli ATCC 25922 was purchased from Iran Research Organization for Science and
technology. MG1655 was a laboratory strain of Prof. R. G. Lloyd.

Media

The liquid medium used for bacterial growth was Luria broth (LB) (Merck,
Germany), and the solid medium was LB supplemented with 1.5% agar (LBA) (Merck,
Germany).

Methods

Antibiotic susceptibility test

MICs of CFX for control strains, ATCC 25922 and MG1655, were determined using
broth dilution method (18). For each strain, two independent cultures were grown
for 24 h at 37?C in LB without CFX. Inocula of 106 colony forming units (CFU)
from each culture were inoculated in duplicate onto screw capped tubes
containing different concentrations of CFX ranging from 5 ng/mL to 50 ng/mL in 8
mL LB broth. The MIC was defined as the lowest concentration of CFX which
prevented any detectable growth after 24 h of incubation at 37?C. MICs for
control strains were determined in three independent experiments. MICs for
CFX-resistant clones isolated during isolation of CFX-resistant mutant (see
below) were measured with the same conditions with higher concentrations of CFX.

Isolation of spontaneous CFX-resistant mutants from solid medium

For each control strains, five independent cultures were grown overnight in LB
without CFX (permissive medium). Viable cell counts in these cultures were
determined by plating several dilutions on LBA without CFX. 150 ?L samples from
each overnight culture (containing approximately 108 cells/mL) were spread in
duplicate on LBA containing CFX (non-permissive medium). At 24 h intervals
(totally for three days) visible colonies were counted and randomly picked up
and cultured for determination of mutation frequency and MIC, respectively.

PCR amplication and DNA sequencing

A single colony from each clone grown on LBA containing CFX was suspended in 100
?L of sterile water and then heated at 95?C for 3 min and cooled on ice. It can
be used as a PCR template for gyrA fragment amplification with the gyrA specific
primers, including forward 5΄-CTGAAGCCGGTACACCGT-3΄ and reverse
5΄-GGATATACACCTTGCCGC-3΄ primers (19). This region of the gyrA gene (577 bp)
contains the QRDR. QRDR is a small region from amino acid 67 to amino acid 106
in GyrA (A subunit) (12).

Results

The MIC of CFX for control strains, including MG1655 (nonpathogenic) and
E. coli ATCC 25922 were determined. They were 35 ng/mL and 9 ng/mL for MG1655 and
E.
coli ATCC 25922, respectively. These figures are consistent with those were
previously obtained (20).

To isolate spontaneous CFX-resistant mutants, MG1655 and
E. coli ATCC 25922 were
grown in permissive liquid medium and then plated on to LBA containing 40 ng/mL
and 12 ng/mL CFX, respectively. The frequency of CFX-resistant mutation for E.
coli ATCC 25922 was 10?10-6 mutant/total counts/day and for MG1655 was 40?10-7
mutant/total counts/day. These are consistent nearly with previous data (20).
Five colonies were randomly selected from each plate, and totally 100
CFX-resistant colonies originated from both control strains were used for
further investigation.

The MICs of CFX for these clones were determined and presented in increasing
order of resistance in Table 1. MICs of majority of clones, which were taken
after a day of incubation and originated from MG1655, were 62.5 or 75 ng/mL
(Table 1). Moreover, most of clones originated from E. coli ATCC 25922 and taken
on the first day of incubation had MIC of 18 ng/mL (Table 1). However, MICs of
clones formed on the third day of incubation were higher than above figures.
This means that these clones may gain extra mutations following exposure to CFX.

On the basis of the MIC results, totally 50 clones from both genetic
backgrounds, considering full range of MIC, were chosen for PCR analysis. Part
of gyrA gene containing QRDR was amplified. Figure 2 shows the result of PCR
amplification. The same results were obtained for all clones. Then, the PCR
products were sequenced by using forward or reverse primer. Finally, these
sequences were compared with the published gyrA sequence of E. coli K-12 by
using DNA for windows software. Putative fluroquinolone resistance mutations
were defined as nucleotide alterations, including substitutions, deletions, or
insertions. The sequence comparison showed that CFX resistance property is
associated with alterations in nucleotide sequence of gyrA gene (Figure 3).
Nucleotide substitutions (Figure 3a, b, c) and deletion (Figure 3d) were seen in
different sites of gyrA gene. These kinds of alterations, shown in Figure 3,
were obtained from different clones.

Alterations in nucleotide sequence of gene would cause a change in the amino
acid sequence of the encoding protein product. Table 2 shows the amino acid
changes that were caused by nucleotide changes shown in Figure 3. Silent
mutations are not presented. Forty five out of fifty clones carried mutations in
gyrA. The remaining might have mutations in other CFX target sites in genes,
such as gyrB, parC or parE.

Of 45 clones harboring mutation in gyrA, 41 carried mutation altering amino acid
83. Amino acid change detected at this site was Ser to Leu. This kind of
mutation has already been reported (14, 15). Moreover, in one out of 4 remaining
clones the amino acid 83 was deleted. This shows the importance of this position
in gain of CFX resistance. Clones containing the alteration of amino acid 83 had
different MICs, suggesting that those with higher MICs probably had extra
mutations in other genes containing CFX target sites. In addition, in two other
non-83 codon alteration clones, there was a mutation that alters Tyr-50 to Phe.
Occurrence of mutation outside of the QRDR region was reported, but it was at
position 51 (17). On the other hand, in the last remaining clone, there was a
mutation which altered Ala-119 to Glu. This codon is near Tyr-122 which is the
active site of GyrA (16). This mutation has not been reported before.

Discussion

The high potency, broad spectrum of activity and relative tolerability of the
fluoroquinolones, like CFX have led to widespread use and misuse of these agents
for different therapeutic purposes. These lead to rapid emergence of resistant
strains to these agents. Fluoroquinolones target enzyme in Gram-negative
bacteria, such as E. coli is DNA gyrase. Mutations in both gyrA and
gyrB
encoding subunits of DNA gyrase lead to resistance to these agents. The majority
of resistant clinical isolates possess mutations in QRDR section of GyrA (14,
15). This study investigated the generation of spontaneous CFX-resistant mutants
in cultures of E. coli strains with different genetic backgrounds.

Our study showed that the frequency of CFX resistant mutants in cultures of
E.
coli strains is low in laboratory condition. However, some of isolated mutants
had high MICs following exposure to CFX. This implies why misuse or
inappropriate use of this drug could lead to rapid development of resistance in
vivo.

We found that most of CFX-resistant mutants contained mutations in QRDR section
of GyrA at amino acid 83 without considering their genetic backgrounds
(pathogenic or non pathogenic). It was suggested that the replacement of
hydrophilic amino acid (Ser) by hydrophobic amino acid (Leu, Trp, Ala or Pro) at
amino acid 83 leads to induction of a local conformation change of the A subunit
(12). This implies the importance of this site in interaction of gyrase and CFX.
Moreover, our finding that showed the deletion of amino acid 83 leads to
resistance to CFX reconfirms the importance of this site and suggests that
either substitution or deletion of this site leads to the loss of enzyme-drug
interaction.

These clones possessed different MICs of CFX. This reveals that the generation
of a mutation in QRDR is the minimum necessity for resistance to CFX and gaining
extra mutations in other CFX targets results in elevation of resistance.
Furthermore, finding two clones with a mutation at position 50 outside the QRDR
region, suggests the importance of more amino acids in the production of active
site of DNA gyrase.

Our finding that showed the alteration of Ala-119 to Glu alone causes resistance
to CFX also suggests that a change from hydrophobic amino acid to acidic amino
acid at amino acid 119 may have an effect on the conformation of the active site
of enzyme (Tyr-122) and its interaction with neighboring amino acids and thereby
the inability of CFX to bind to gyrase. In addition, for this clone having the
same MIC as most of those with substitution at Ser-83 (75 ng/mL) indicates that
position 119 may be as important as position 83 in making bacteria resistant to
CFX.

Briefly, the presence of mutations at amino acids 50 and 119 suggests that in
addition to QRDR section and Tyr-122, these sites are also essential for DNA
gyrase activity.

Acknowledgements

This work was financially supported by the University of Shahrekord. We thank
Prof. R. G. Lloyd for kind gift of MG1655. We also acknowledge Genfanavaran
Company for providing us with primers and sequencing facility.